This proposal combines molecular genetics with the use of reconstituted cell-free systems to probe the mechanisms by which newly synthesized proteins are localized to their correct subcellular compartments and by which newly synthesized integral membrane proteins achieve their correct transmembrane orientation. We will create a systematic serie of gene deletions, insertiosn and point mutations in signal and stop transfer sequences, in order to identify their functional regions upon expression in cell-free reconstituted and whole cell systems. By engineering the implicated functional gene segments into the coding regions for other proteins we will study the rules by which protein orientation and localization proceed. Attempts will be made to extend our approach to other classes of localization events beyond the endoplasmic reticulum, and efforts will be directed towards using our family of mutants to identify and purify receptor components involved in secretion and membrane assembly. Through this work a more precise understanding of the molecular events of intracellular protein topogenesis will emerge and a "topogenic library" will be established with which to redirect proteins to locations and orientations of choice.